The ubiquitin-proteasome system regulates many essential cellular processes including cell division. The recent development of cancer drugs that inhibit the proteasome suggests that the ubiquitin-proteasome system may provide many new therapeutic targets. The goal of this project is to develop new chemical inhibitors of the ubiquitin-proteasome system, and to understand how this pathway regulates cell division. We first plan to characterize novel small molecule inhibitors of the Anaphase-Promoting Complex (APC), a ubiquitin ligase that is essential for cell division. APC inhibitors may be useful for the treatment of cancer, because they may inhibit cell division or sensitize cells to antimitotic drugs such as taxol. Additionally, APC inhibitors may be useful for the treatment of diseases that involve axonal injury, because the APC negatively regulates axon growth. Our studies of these small molecules will also provide important new insights into how the APC is regulated by activator proteins. Another goal of our studies is to characterize ubistatins, compounds that prevent ubiquitin- dependent proteolysis by binding to ubiquitin chains. However, the mechanism of ubiquitin chain binding remains unclear, and we plan to synthesize new ubistatin derivatives that will allow us to determine the structure of ubistatins bound to ubiquitin chains. The structural information will enable us to synthesize more potent and perhaps more cell-permeable versions of ubistatins, enhancing the utility of these compounds to investigators in the field, and accelerating the development of ubiquitin-binding compounds as drug candidates. A third goal of our research program is to understand how ubiquitinated substrates are recognized for degradation by the proteasome. It has been assumed that a specific type of ubiquitin chain (linked through lysine-48 of ubiquitin) is required for degradation by the proteasome. Our studies of ubiquitinated cyclin B1 suggest that other types of ubiquitin linkages may be sufficient to target the protein for degradation. We therefore plan to determine the types of ubiquitination that are required for degradation of cyclin B1 by purified proteasomes and in complex cell extracts. These studies are critical for understanding how small molecule inhibitors of ubiquitination and ubiquitin chain recognition may perturb degradation. Finally, we want to understand how the localization of substrates affects their degradation by the ubiquitin-proteasome system. We have identified a novel sequence element in cyclin B1 that recruits the protein to chromatin during mitosis. We plan to identify the protein that recruits cyclin B1 to chromatin, and to determine the functional consequences of cyclin B1 association with chromatin. Together these studies will provide new chemical tools for studying the ubiquitin-proteasome pathway, and will help us understand how a critical substrate of this pathway, cyclin B1, is regulated during the cell cycle.